The essential role of selenium in nutrition was first recognized by Schwarz and Foltz in 1957 (Schwarz, K. and Foltz, C. M., Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Soc. 79:3292 (1957)). These researchers observed that rats developed liver necrosis when fed a purified diet deficient in vitamin E. However, the addition of selenium to the diet prevented the development of this condition. The ability of dietary selenium to prevent the development of exudative diathesis, a condition characterized by leakage of plasma into subcutaneous spaces of the abdomen and breast in chicken, was reported in the same year by Patterson et al (Patterson, E. L., Milstrey, R., Stokstad, E. L. R. Effect of selenium in preventing exudative diathesis in chicks. Proc. Soc. Exp. Biol. Med. 95: 617-620 (1957)). The important role of selenium in nutrition was further demonstrated by recognizing the practical effect of selenium deficiency in livestock (Muth, O. H., Oldfield, J. E., Remmert, L. F., and Schubert, J. R. Effects of selenium and vitamin E on white muscle disease. Science 128: 1090 (1958) and Hartley, W. J., and Grant, A. B. A review of selenium responsive diseases of New Zealand livestock. Fed. Proc. 2o: 679 (1961)). Subsequent work confirmed that selenium is an essential element for animals and that its deficiency results in various disorders (Combs, G. F. Jr., Combs, S. B. The role of selenium in nutrition. Academic Press, Orlando, Fla., pp 265-399 (1986b)).
The importance of selenium in human nutrition and the effects of its deficiency on human health were not recognized until the 1970s. Selenium deficiency was found to be one of the factors responsible for the Keshan disease, a human condition characterized by a dilated cardiomyopathy that affects persons living in rural areas of China. The incidence of the Keshan disease matched the distribution of selenium-deficient areas (Keshan Disease Research Group of the Chinese Academy of Medical Sciences. Epidemiologic studies on the etiologic relationship of selenium and Keshan disease. Chin. Med J. 92:477-482 (1979)). Furthermore, a prospective placebo-controlled study demonstrated that new cases of the disease can be prevented by the administration of sodium selenite tablets (Keshan Disease Research Group of the Chinese Academy of Medical Sciences. Observations on effect of sodium selenite in prevention of Keshan disease. Chin. Med J. 92:471-477 (1979)). The detrimental effects of diet-induced selenium deficiency in critically ill patients were reported in several case studies. Skeletal myopathy developed in one patient on total parenteral nutrition and was reversed by intravenous administration of selenomethionine (van Rij, A. M., Thomson, C. D., McKenzie, J. M., Robinson, M. F. Selenium deficiency in total parenteral nutrition. Am. J. Clin. Nutr. 32: 2076-2085 (1979)). Fatal cardiomyopathy induced by nutritional selenium deficiency was reported in a 43-year-old man receiving parenteral alimentation for 2 years before his death (Johnson, R. A., Baker, S. S., Fallon, J. T., Maynard, E. P., Ruskin, J. N., Wen, Z., Ge, K., and Cohen, H. J. An occidental case of cardiomyopathy and selenium deficiency. The New England Journal of Medicine. 304: 1210-1212 (1981)). In 1982, a second case of fatal cardiomyopathy associated with dietary selenium deficiency was reported in a patient on home parenteral nutrition for at least two years (Selenium Deficiency and Fatal Cardiomyopathy in a Patient on Home Parenteral Nutrition. Gastroenterology. 83:689-693 (1982)).
The recognition of the essential role of selenium in human and animal nutrition has resulted in the establishment of a Recommended Daily Allowance (RDA) for humans and approval of the inclusion of additional selenium compounds in animal feed. Recently, the Food and Nutrition Board of the Institute of Medicine revised the RDA for selenium to 55 μg (Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. Washington, D.C.: National Academy Press, (2000)). In 1974, the Food and Drug Administration (FDA) approved sodium selenite and sodium selenate as feed additive. These inorganic selenium salts can be added at the level of 0.3 ppm Se in feed dry matter. In June 2000, the FDA approved the use of selenium yeast in poultry broiler and layer diets.
The biochemical mechanism involved in manifesting the beneficial effects of selenium began to emerge in 1973 when selenium was found to be an essential component of the antioxidant enzyme glutathione peroxidase (Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G. F., and Hockstra, W. G. Selenium: Biochemical Role as a Component of Glutathione Peroxidase. Science, 179: 588-590 (1973) and Flohe, L., Gunzler, W. A. and Shock, H. H. Glutathione Peroxidase. A Selenoenzyme. FEBS Lett. 32: 132-134)). Concurrently, an extra cellular selenoprotein (Selenoprotein P) was discovered in rat, rhesus monkey and human plasma and found to be different than glutathione peroxidase. Selenoprtoein P is a monomeric glycoprotein that contains about 380 amino acid residues and up to 10 selenocysteine residue per the polypeptide chain. It is ubiquitous in mammalian tissues and accounts for more than 40% of the selenium concentration in human plasma. In human plasma in vitro, Selenoprotein P protects against peroxynitrite-mediated oxidation and reduces phospholipids hydroperoxide suggesting that it functions as an antioxidant in vivo. (Moschos M. P. Selenoprotein P. Cellular and Molecular Life Sciences. 57: 1836-1845 (2000)). Another function of selenium is as a catalytically active component of the iodothyronine deiodinase enzymes that regulates thyroid hormone metabolism. More recently, selenocysteine was identified in the active center of thioredoxin reductase demonstrating the role selenium plays in various metabolic processes catalyzed by these enzymes.
Recent studies have shown that the role of selenium in mammalians is not limited to the physiological functions of selenoenzymes. It now appears that selenium has a very specific role in spermatogenesis that is essential for male fertility. The selenoprotein phospholipids hydroperoxide glutathione peroxidase was found to play important roles during sperm maturation (Ursini F., Heim S., Kiess. M., Maiorino M., Roveri A., Wissing J., Flohe' L. Dual Function of the Selenoprotein PHGPx During Sperm Maturation. Science 285: 1393-1396 (1999)). The identification of a specific selenoenzyme in the sperm nuclei further underscored the important role selenium plays in sperm maturation (Pfeifer H., Conrad M., Roethein D., Kyriakopoulos A., Brielmeier M., Bornkamm G. W., Behne D. Identification of a Specific Sperm Nuclei Selenoenzyme Necessary for Protamine Thiol Cross-Linking During Sperm Maturation. FASEB J 15: 1236-1238 (2001)).
Selenium is present in selenoproteins in the form of selenocysteine. All mammalian selenoproteins identified so far are enzymes in which the selenocysteine residue is responsible for their catalytic function. Further studies of the complete amino acid sequence of mammalian glutathione peroxidase and the DNA analysis of its gene revealed that a specific codon is responsible for the insertion of selenocysteine into the amino acid chain of these enzymes. These results demonstrated that selenocysteine is the 21st encoded amino acid. This insertion is directed by a specific codon in mRNA. This codon is recognized by the anticodon of a specific tRNA which is first loaded with serine. The replacement of the side-chain oxygen of serine with selenium is catalyzed by a selenocysteine synthase to convert the seryl-tRNA into selenocysteyl-tRNA. Selenophosphate produced from selenide and ATP serves as the selenium donor in this reaction. Selenocysteine from selenocystyl-tRNA is incorporated into the selenoprotein at the ribosome.
Labeling rats and mice with 75Se indicated the presence of numerous selenium-containing proteins in mammalian tissues. Most importantly, a hierarchy in the distribution of selenium in the various tissues was discovered. This hierarchy ensures the preferential supply of selenium to certain proteins in certain organs and further illustrates the biological significance of selenium. When selenium intake is insufficient, the levels of selenium in some tissues such as the brain, the endocrine and the reproductive organs are maintained by preferential supply of dietary selenium and redistribution of the metabolized element. Extreme selenium depletion in rats resulted in a drastic decrease in selenium levels in most tissues to 1-3% control levels except for the brain which retained 60% of control level followed by spinal marrow, pituitary, thyroid, ovaries and adrenals (Behne D., Pfeifer H., Rothlein D., Kyriakopoulos A. Cellular and Subcellular Distribution of Selenium and Selenium-containing proteins in the rat. In: Roussel A. M., Favier A. E., Anderson R. A. (eds) Trace Elements in Man and Animals 10, Kluwer Academic/Pllenum Publishers New York, pp 29-34 (2000)). A further proof of the importance of selenium in mammalian is demonstrated by the finding that disrupting selenoprotein synthesis by knocking out the gene for selenocystyl-tRNA was lethal at the embryonic stage in mice. In contrast, severe selenium deficiency did not produce similar lethal effects probably due to the hierarchy in selenium distribution.
The dietary requirements for selenium are usually fulfilled by the ingestion of diets containing naturally occurring organic selenium compounds. Food and feed ingredients rich in organic selenium compounds include meat, fish, dairy products, some vegetables and grains. The concentration of selenium in materials of plant origin often depends on the concentration of selenium in the soil where the plants were grown. The soil of the Rocky Mountain States contains higher levels of selenium than other states and plants growing on these soils contain higher levels of selenium. The majority of organic selenium in natural food and feed ingredients is present as L-selenomethionine. Some accumulator plants and vegetables such as garlic, onions and broccoli growing on selenium rich soils contain Se-methylselenocysteine and its derivatives as the major organic selenium compounds. One of the predominant forms of selenium in native forage plants of the U.S. is selenate. Of 24 plants studied, selenate represented 5-92% of total selenium. Selenite was absent in all but one of these plants which contained 3% of total selenium as selenite. (Whanger P. D. Selenocompounds in Plants and Animals and their Biological Significance. Journal of the American College of Nutrition, 12: 223-232 (2002)). Regardless of the form in which the selenium is ingested, it is transformed by a variety of metabolic pathways via the same intermediary pool into the specific selenocysteine-containing selenoproteins which are responsible for selenium biological effects. The levels of these selenocysteine-containing selenoproteins in tissues appear to be homeostatically controlled. Ingestion of supplemental selenium above the optimal requirements does not appear to increase the concentrations of the specific selenoproteins in tissues. However, ingestion of selenomethionine results in higher retention of selenium in tissues than those observed with other sources of selenium. This is attributed to the fact that only a fraction of selenomethionine is metabolized similar to other sources of selenium via the intermediary pool to specific selenocysteine-containing proteins. A certain percentage of ingested selenomethionine is incorporated non-specifically directly into proteins in place of methionine. This non-specifically bound selenium is present in high concentrations in methionine rich proteins. The fraction of ingested selenomethione that is incorporated in non-specific proteins appears to be dependent on the ratio of selenomethionine to methionine and not selenium status. When low methionine diets are ingested, the increased non-specific incorporation of selenomethionine in proteins resulted in the decreased concentrations and effects of the specific selenoproteins. Non-specific incorporation of selenomethionine takes place in the proteins of skeletal muscles, erythrocytes, pancreas, liver, stomach, kidneys and the gastrointestinal mucosa. The release of selenomethionine from body proteins is linked to protein turnover. A steady state concentration of selenomethionine in tissues may be established if the intake of the seleno-amino acid is maintained over extended period of time. (Schrauzer G. N. Nutritional Selenium Supplements: Product Types, Quality, and Safety. Journal of the American College of Nutrition, 20: 1-4 (2001)).
The disposition of selenomethionine, Se-methyl-selenocysteine, selenite, and selenate in animals has been carefully studied. These common sources of selenium in animal nutrition take different pathways to the intermediary selenium pool which is ultimately incorporated in the specific seleno-proteins or further converted into polar metabolites that can be readily excreted. Below, these pathways are briefly described.
A fraction of ingested selenomethionine is converted to selenocysteine by the normal biochemical pathway involved in converting methionine to cysteine. Se-Adenosyl-selenomethionine is formed from selenomethionine and adenosine by a reaction catalyzed by S-Adenosylmethionine synthase. The conversion of Se-Adenosyl-selenomethionine to Se-adenosyl-selenohomocysteine is catalyzed by specific methyl transferases. The enzyme S-Adenosyl-homocysteine lyase converts Se-adenosyl-selenohomocysteine to selenohomocysteine. The addition of selenohomocysteine to serine to form Se-cystathionine is catalyzed by cystathionine synthase, a PLP enzyme. Se-Cystathionine is converted to selenocysteine, ammonia and 2-ketobutyric acid in a reaction catalyzed by Cystathionine-γ-lyase. The enzyme Cysteine-β-lyase converts selenocysteine into hydrogen selenide, pyruvic acid and ammonia. Hydrogen selenide (H2Se) is the key intermediary metabolite involved in the biosynthesis of specific seleno-proteins. The enzyme selenophosphate synthase catalyzes the reaction between hydrogen selenide and ATP to produce the reactive selenium compound selenophosphate (H3SePO3), AMP and orthophosphate. A specific enzyme, selenocysteine synthase then catalyzes the replacement of the side-chain oxygen of serine in a unique seryl-tRNA to give selenocysteyl-tRNA. Selenocysteine is transferred from selenocysteyl-tRNA and inserted in the forming selenoprotein chain on the ribosome.
Selenite is partially reduced to elemental selenium by dietary constituents such as ascorbic acid and by bacterial flora in the gastrointestinal tract. Absorbed selenite is readily reduced in two steps by four equivalents of glutathione to selenoglutathione trisulfide. The selenoglutathione trisulfide is reduced by glutathione in two steps to give hydrogen selenide and two moles of oxidized glutathione. hydrogen selenide serves as selenium source for the biosynthesis of selenoproteins as described above. If selenate is ingested as the selenium source, it is first reduced by two equivalents of glutathionine to selenite which is then converted to hydrogen selenide.
A fraction of the ingested selenium source is eliminated via a number of pathways. Some of orally ingested selenite and selenate is reduced in the gastrointestinal tract to elemental selenium which is excreted in feces. Selenite and selenate are also excreted in urine. Enzymatic methylation of hydrogen selenide gives the volatile metabolite methyl selenol which can be excreted by the lungs. Methyl selenol is further methylated in a stepwise fashion first to the volatile metabolite dimethyl selenide which is excreted by the lungs and then to the polar trimethylselenonium cation which is eliminated in the urine.
Supplementation of animal feed with an approved source of selenium is gaining popularity. Currently, inorganic sources such as selenite and selenate as well as the organic source selenium yeast are approved by the FDA as feed ingredients. However, the amount of selenium that can be added and the species of livestock that may be supplemented are regulated. The approval of the use of the inorganic sources of selenium such as selenite and selenate as feed ingredients is curious since these do not occur naturally in significant concentrations in feed. L-Selenomethionine is the form of selenium most commonly present in natural foods and feed. However, synthetic L-selenomethionine has not been commercially available at reasonable prices for use as feed ingredient in livestock production. Therefore, selenium enriched yeast has been used as a practical affordable source of L-selenomethionine. Special strains of Saccharomyces cerevisiea grown in a selenium rich medium accumulate as much as 3000 μg Se per g dry matter. Most of the selenium in yeast exists as L-selenomethionine. The L-selenomethionine is present primarily incorporated in the yeast protein in place of L-methionine. Other organic selenium compounds may be present in low concentrations including Se-adenosyl-selenohomocysteine (2-5%), selencysteine (0.5%), methylselenocysteine (0.5%), selenocystathionine (0.5%), and γ-glutamyl-Se-methylselenocysteine (0.5%). Only traces of inorganic selenium may be present in the yeast as selenite or selenate (Schrauzer G. N. Selenomethionine: A Review of its Nutritional Significance, Metabolism and Toxicity. J. Nutr. 130: 1653-1656 (2000)).
Several studies were published during the last several years comparing the effects of selenite and selenium yeast supplements on the selenium status and health of livestock. Especially in selenium deficient animals, the selenium concentrations in plasma and tissues increase linearly as intake of selenium increases to a point after which plasma and tissue selenium concentrations do not change significantly with increased intake. For example the relationship of dietary selenium from sodium selenite to selenium concentrations in plasma and milk in dairy cows was examined by Maus et al. Selenium concentration in plasma and milk increased linearly as intake of selenium increased from about 2-6 mg/day. Further increases in intake resulted in only little change in plasma and milk selenium (Maus R. W., Martz F. A., Belyea R. L. and Weiss M. F., Relationship of Dietary Selenium to Selenium in Plasma and Milk from Dairy Cows, J Dairy Sci, 63: 532-537-(1980)).
Selenium was found to be more bioavailable from selenium yeast than from selenite or selenate in several animal studies. The increase in tissue selenium concentration was greater in animals fed selenium yeast compared to animals fed selenite. However, the increase in glutathione peroxidase activity was about the same regardless of the source of supplemental selenium. The favorable effects of selenium supplementation on animal health were demonstrated in several studies. For example, selenium supplementation improved udder health in dairy cows as demonstrated by a decrease in the percent quarters harboring mastitis pathogens and a decrease in somatic cells count in milk. Again the effects of selenium yeast were greater than those of sodium selenite (Malbe M., Klassen M., Fang W., Mylls V., Vikerpuur M., Nyholm K., Sankari S., Sourta K., and Sandholm M. Comparisons of Selenite and Selenium Yeast Feed Supplements on Se-incorporation, Mastitis and Leucocyte Function in Se-deficient Dairy Cows, J Vet Med A, 42: 111-121 (1995)).
It is now well established that dietary selenium is essential for the health and wellbeing of humans and animals. Several studies have demonstrated that selenium is more bioavailable from organic sources than from inorganic sources. The only organic selenium source available for commercial use is selenium rich yeast preparation. In yeast, selenium exists primarily as L-selenomethionine rich proteins. Although Selenium yeast is now widely accepted as a source of dietary selenium, its use suffers from several shortcomings. The concentration of organically bound selenium in yeast is limited by its ability to form L-selenomethionine from the selenite enriched media. Currently, the highest possible concentration of selenium in yeast appears to be 2000 μg/g dry matter. Secondly, since the organically bound selenium in yeast is produced by a biological process that is vulnerable to subtle variations in the large scale production process, the exact composition of the selenium compounds is variable and is not readily known. Occasionally, yeast contains variable concentrations of inorganic selenium compounds such as selenites and selenates. Thirdly, the organic selenium compounds are present in yeast as part of the intracellular proteins. Before these compounds are available for absorption after being ingested, the cell walls of yeast must rupture to release the protein into the animals' gastrointestinal tract where it can be subjected to the proteolytic effects of digestive enzymes. It is only after the protein is hydrolyzed to single amino acids or dipeptides that the selenium compounds can be absorbed. The release of the selenium compounds as single amino acids or dipeptides from the intact yeast cells is not complete and is highly dependent on the conditions in the gastrointestinal tract. Because of these shortcomings, it is important to develop alternatives to selenium enriched yeast to serve as a readily bioavailable dietary source of selenium.
Recently, synthetic seleno-amino acids became commercially available at a reasonable cost. These amino acids have low water solubility and their crystals have water repellent properties that result in low rate of dissolution. Low solubility and slow rate of dissolution lower the bioavailability of these compounds after feeding to animals.
Recently, the demand for a dietary sources of selenium with improved bioavailability for use as a supplement for human and livestock. has increased. Synthetic seleno-amino acids have recently become commercially available at a reasonable cost. These amino acids have low water solubility and their crystals have water repellent properties that result in low rate of dissolution. Low solubility and slow rate of dissolution lower the bioavailability of these compounds after feeding to animals. One objective of this invention is to identify derivatives of seleno-amino acids with improved bioavailability.
The inventors of this patent application have in the past disclosed 1:1 complexes of essential metals and amino acids as means of improving the bioavailability of these metals after ingestion in livestock, see for example U.S. Pat. Nos. 3,941,818, 3,925,433, 3,950,372, 4,021,569, and 4,067,994. The purpose of forming these complexes is to increase the solubility of the metal and increase its stability in chemical environment of the gastrointestinal tract of the animal. Although the increased bioavailability of the trace metal was anticipated and consistently demonstrated in numerous studies, the increased bioavailability of the amino acid ligand was not anticipated. Because of its unique chemical properties, selenium in contrast to the essential metals previously described in our patents does not form complexes with amino acids. Therefore, it was necessary to find a different strategy to improve the bioavailability of selenium.
Selenium like sulfur, is a member of group VIA elements. It exists in different allotropic forms and has oxidation states of −2, 0, +2, +4, and +6. Selenium is a nonmetallic element. It can form mono-atomic anions and therefore can form ionic as well as covalent bonds. In the oxidation state −2, selenium forms covalent bonds with carbon substituents and can often replace sulfur in naturally occurring compounds. The biological role of selenium is attributed to these naturally occurring compounds in which selenium exists in the −2 oxidation state and is covalently bound, usually with carbon as part of functional proteins. Seleno-amino acids have been proposed as dietary sources of selenium. However, it is recognized that the bioavailability of these compounds may be significantly diminished by the nutritional status of the animal and the composition of the diet and gastrointestinal tract contents. Therefore it was desirable to explore reversible derivatives of the seleno-amino acids that may improve the bioavailability of these amino acids. The improved bioavailability of essential metals from the 1:1 amino acid complexes did not provide any knowledge on the relative bioavailability of the amino acid ligands. Indeed, the common knowledge that amino acid are absorbed from the gastrointestinal tract by a highly specialized mechanisms indicated that the formation of these complexes will not have a significant effect on the bioavailability of the amino acid. Therefore it was surprising that these complexes demonstrated improved bioavailability of the seleno-amino acid when used as a ligand.
The primary object of the present invention is to make novel 1:1 metal complexes of seleno-amino acids with improved bioavailability.
Another object of the invention is to describe methods of preparation of these derivatives and their use as feed ingredients in livestock.